Summary of Work: More than 1 in 4,000 children born in the United States each year will develop a mitochondrial disease by age 10 with a mortality rate from 10 to 50 percent. Defects in mitochondrial function have been linked to several of the most common diseases of aging. Over 50 million people in the US suffer from chronic degenerative disorders involving mitochondria of compromised function. The mutation rate of the mitochondrial genome is 10-20 times greater than in the nuclear DNA and is 16 times more prone to oxidative damage than nuclear DNA. Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase g (pol g), we previously investigated the fidelity of DNA synthesis by human pol g. Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mitochondrial DNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase g. Since Y955 is a highly conserved residue critical for nucleotide recognition among Family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (kcat) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (KM). The Y955C derivative is twofold less accurate for base-pair substitutions than wild-type pol g despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C pol g is consistent with the accumulation of mtDNA mutations in patients with PEO. The mitochondrial respiratory chain is a source of endogenous reactive oxygen species (ROS), and oxidative modification of biomolecules, including proteins, can alter their normal functions. Since pol gamma is associated with DNA within the mitochondrial matrix, this enzyme is subject to oxidation in vivo by hydrogen peroxide and iron ions associated with mtDNA. The effect of H2O2 on the enzymatic activities and DNA binding efficiency of pol gamma has been examined. Hydrogen peroxide inhibits the DNA polymerase activity of the p140 subunit and lowers its DNA-binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H2O2 treatment offers protection from oxidative inactivation. Pol gamma can be detected as one of the major oxidized proteins in the mitochondrial matrix, and the degree of oxidation correlates with a decline in polymerase activity. These results suggest that pol gamma is a target for oxidative damage by ROS which may impair mitochondrial DNA replication and repair.